Comparative characterization of STRO-1(neg)/CD146(pos) and STRO-1(pos)/CD146(pos) apical papilla stem cells enriched with flow cytometry

Abstract

Objective: Stem Cells residing in the Apical Papilla (SCAP) of human permanent teeth represent a promising cell source for dental tissue regeneration. Therefore, the functional and molecular properties of specific subpopulations existing within heterogeneous cultures should be further investigated to give insight whether their selection could be beneficial for targeted therapeutic applications.

Design: In this study we extensively characterized SCAP cultures established from 10 healthy subjects, as well as their STRO-1(pos/)CD146(pos) and STRO-1(neg/)CD146(pos) subpopulations isolated with fluorescence-activated cell sorting. SCAP were analyzed for embryonic (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin) and hematopoietic (CD117/c-kit, CD45) stem cell (SC) markers using flow cytometry. Multipotentiality was evaluated with culture specific staining (Alizarin-Red-S, Oil- Red-O) and RT-PCR analysis for osteo/odontogenic (DSPP, BSP, ALP, osteocalcin, osteonectin, BMP-2, Runx2), adipogenic (lipoprotein-lipase-LPL) and neurogenic (Neurofilament/NFL-L, nestin, β-tubulin-III, NCAM) markers.

Results: Our results showed that the STRO-1(pos)/CD146(pos) subpopulation demonstrated higher CFU efficiency and much higher expression of several embryonic and mesenchymal SC markers compared to the non-sorted SCAP. They also showed enhanced odontogenic differentiation potential, as evidenced by higher mineralization capacity and expression of osteo/odontogenic markers. By contrast, absence of STRO-1 in the STRO-1(neg)/CD146(pos) subpopulation yielded the opposite results and was associated with significant downgrading of the above-mentioned properties.

Conclusions: These results suggest that STRO-1(pos)/CD146(pos) SCAP cells represent a very promising adult MSCs source with enhanced multipotent SC properties that could be easily isolated with simple flow cytometric methods to be used for tissue engineering applications.

Keywords: Apical papilla stem cells (SCAP); CD146 perivascular antigen; Embryonic stem cell markers; Fluorescence-activated cell sorting; Multilineage differentiation potential; STRO-1 antigen.